Abstract

Insulin stimulation of phosphatidylinositol (PI) 3-kinase activity is defective in skeletal muscle of type 2 diabetic individuals.
We studied the impact of antidiabetic therapy on this defect in type 2 diabetic subjects who failed glyburide treatment by
the addition of troglitazone (600 mg/day) or metformin (2,550 mg/day) therapy for 3–4 months. Improvement in glycemic control
was similar for the two groups, as indicated by changes in fasting glucose and HbA1c levels. Insulin action on whole-body glucose disposal rate (GDR) was determined before and after treatment using the hyperinsulinemic
(300 mU · m−2 · min−1) euglycemic (5.0–5.5 mmol/l) clamp technique. Needle biopsies of vastus lateralis muscle were obtained before and after each
3-h insulin infusion. Troglitazone treatment resulted in a 35 ± 9% improvement in GDR (P < 0.01), which was greater than (P < 0.05) the 22 ± 13% increase (P < 0.05) after metformin treatment. Neither treatment had any effect on basal insulin receptor substrate-1 (IRS-1)-associated
PI 3-kinase activity in muscle. However, insulin stimulation of PI 3-kinase activity was augmented nearly threefold after
troglitazone treatment (from 67 ± 22% stimulation over basal pre-treatment to 211 ± 62% post-treatment, P < 0.05), whereas metformin had no effect. The troglitazone effect on PI 3-kinase activity was associated with a 46 ± 22%
increase (P < 0.05) in the amount of the p110β catalytic subunit of PI 3-kinase. Insulin-stimulated Akt activity also increased after
troglitazone treatment (from 32 ± 8 to 107 ± 32% stimulation, P < 0.05) but was unchanged after metformin therapy. Protein expression of other key insulin signaling molecules (IRS-1, the
p85 subunit of PI 3-kinase, and Akt) was unaltered after either treatment. We conclude that the mechanism for the insulin-sensitizing
effect of troglitazone, but not metformin, involves enhanced PI 3-kinase pathway activation in skeletal muscle of obese type
2 diabetic subjects.

Resistance to the effects of insulin on glucose uptake and metabolism in skeletal muscle is a major contributor to the pathogenesis
of insulin-resistant states, such as obesity and type 2 diabetes (1,2). Intense interest has focused on identifying the steps in the insulin-signaling network that are responsible for the stimulation
of glucose transport and that might be defective in insulin-resistant states. Data indicate that activation of phosphatidylinositol
(PI) 3-kinase is a necessary, albeit not sufficient, step for insulin-induced glucose transport (3–6). Defective stimulation of PI 3-kinase activity by insulin has recently been identified in skeletal muscle of type 2 diabetic
subjects (7,8).

Thiazolidinediones (TZDs) are a new class of insulin-sensitizing agents being used for the treatment of type 2 diabetes (9). The molecular targets of these compounds are thought to include the nuclear receptor peroxisome proliferator-activator
receptor-γ (PPAR-γ), which regulates the expression of numerous genes involved in glucose and lipid metabolism (10). Evidence suggests that TZDs ameliorate insulin resistance and improve insulin-stimulated glucose disposal in skeletal muscle
of type 2 diabetic subjects (11), but their exact mechanism of action remains unclear.

Several lines of evidence support the notion that TZDs enhance insulin signaling. TZDs have been shown to increase insulin-induced
PI 3-kinase and Akt activation in cultured human skeletal muscle cells in vitro (12), with parallel increases in insulin-stimulated glucose uptake and glycogen synthesis (12). In addition, TZDs can prevent and reverse hyperglycemia-induced insulin resistance of insulin receptor and insulin receptor
subtrate-1 (IRS-1) phosphorylation in fibroblasts (13). In vivo in obese Zucker rats, TZDs normalize the phosphorylation of the skeletal muscle insulin receptor (14). Taken together, these data suggest that the effects of TZDs to enhance insulin signaling could be important for their antidiabetic
action. However, to date, the effect of TZDs on insulin signaling in vivo in insulin target tissues of humans has not been
reported.

Metformin is a member of the biguanide class of compounds, which are also effective at lowering glucose concentrations in
patients with type 2 diabetes (15). A number of studies have demonstrated that metformin inhibits gluconeogenesis, reduces hepatic glucose output, and lowers
fasting blood glucose concentration (11,16). In addition to its effect on the liver, metformin has also been reported to decrease glucose concentrations by increasing
peripheral insulin sensitivity and augmenting insulin-mediated glucose uptake in skeletal muscle of type 2 diabetic subjects
(17). The precise mechanism for this action of metformin is incompletely understood, but in vitro studies indicate it could involve
multiple effects, including increased translocation of GLUT1 and GLUT4 glucose transporters from intracellular vesicles to
the cell surface (18) and increased binding of insulin to cell-surface insulin receptors (19). Whereas recent studies have shown that metformin can normalize insulin receptor tyrosine phosphorylation and PI 3-kinase
and Akt activities in adipocytes exposed for long periods to high insulin levels in vitro (20), the effects of metformin on insulin signaling in human or rodent skeletal muscle are still unknown.

The present study was designed to determine whether the insulin-sensitizing effects of troglitazone or metformin could involve
reversal of the defect in insulin-stimulated PI 3-kinase activation in muscle of obese type 2 diabetic subjects. In this study,
we demonstrate that 3 months of troglitazone treatment in obese type 2 diabetic subjects significantly increases insulin-stimulated
PI 3-kinase and Akt activities in skeletal muscle. However, this improvement in insulin signaling is not seen in subjects
treated with metformin, despite similar improvement in blood glucose control. Our data suggest that the mechanism for the
insulin-sensitizing effect of troglitazone, but not metformin, could involve enhanced PI 3-kinase pathway activation in skeletal
muscle of obese type 2 diabetic subjects.

RESEARCH DESIGN AND METHODS

Human subjects and treatment protocol

A total of 14 type 2 diabetic subjects (11 men and 3 women between the ages of 30 and 70 years) who were poorly controlled
(HbA1c >8.5% and fasting plasma glucose >140 mg/dl) on at least half-maximal doses of any sulfonylurea agent were recruited for
these studies. Except for diabetes, the subjects were healthy and on no other medications known to influence glucose metabolism.
After screening, their current sulfonylurea medication was discontinued and all subjects were uniformly started on glyburide
at 10 mg b.i.d. for at least 4 weeks. Baseline studies were subsequently performed and subjects randomized to the addition
of either troglitazone or metformin to glyburide therapy. Subjects were counseled at 2-week intervals to consume a weight-maintenance
diet for the duration of the study. Over 4–6 weeks the troglitazone treatment was titrated up to 600 mg/day or metformin up
to 2,550 mg/day as required to achieve glycemic goals. After 3–4 months of this additional therapy, patients were readmitted
for repeat studies. These subjects were part of a larger study group comparing the effects of troglitazone and metformin therapy
on multiple clinical parameters (21). With regard to clinical parameters and response to treatment, the current subjects were indistinguishable from the larger
study group. The experimental protocol was approved by the Committee on Human Investigation, University of California, San
Diego. Informed written consent was obtained from all subjects after explanation of the protocol.

Protocol

All subjects were admitted to the Special Diagnostic and Treatment Unit at the VA San Diego Healthcare System. To quantitate
peripheral insulin action, all subjects underwent a 3-h hyperinsulinemic (300 mU · m−2 · min−1) euglycemic (5.0–5.5 mmol/l) clamp after a 12-h overnight fast, as previously described (22). The glucose disposal rate (GDR) was determined during the last 30 min of the clamp. Percutaneous needle biopsies of vastus
lateralis muscle were performed before and after 3 h of insulin infusion, as previously described (22), and muscle tissue was immediately frozen in liquid nitrogen. Plasma glucose and insulin levels were determined before each
biopsy.

Determination of PI 3-kinase and Akt activity

Muscle lysates (150 μg protein) were subjected to serial immunoprecipitations with 5 μl of a polyclonal IRS-1 antibody (1:100
dilution; gift from Dr. Morris White, Joslin Diabetes Center) coupled to protein A-sepharose beads (Sigma, St. Louis, MO)
overnight, followed by 2 μg of a polyclonal Akt antibody that recognizes both Akt1 and Akt2 (Upstate Biotechnology, Lake Placid,
NY) coupled to protein G-sepharose beads (Pharmacia Biotech, Piscataway, NJ) for 4 h. The immune complex was washed, and PI
3-kinase and Akt activities were determined as previously described (7). The effect of insulin to stimulate Akt activity tended to be lower than in our previous report (7), probably because of a different immunoprecipitation methodology. In our previous report, we performed immunoprecipitation
for 4 h and obtained approximately a twofold insulin stimulation (300 mU insulin · m−2 · min−1 for 3 h) of Akt activity over the basal value. Because of limitations in the size of muscle samples in the present study,
we carried out serial immunoprecipitations (see above), which could result in less activation of Akt in response to insulin.

Determination of IRS-1, p85, p110β, and Akt1/2 protein amounts

A total of 10 μg tissue lysate protein per lane was resolved by SDS-PAGE (7.5 and 10% gels) and transferred to nitrocellulose
membranes (Schleicher & Schuell). The membranes were blocked with 5% nonfat dry milk overnight at 4°C and incubated with the
following antibodies for 3 h at room temperature: a polyclonal IRS-1 antibody (Upstate Biotechnology), a polyclonal antibody
against the p85α subunit or p110β subunit of PI 3-kinase (Upstate Biotechnology), and a polyclonal Akt antibody (Upstate Biotechnology)
in 1% nonfat dry milk. The membranes were washed and bands visualized as previously described (23).

Statistical analysis

Data are presented as the mean ± SEM. Statistical analyses were performed using the Stat View program (Abacus Concepts, Berkeley,
CA). Statistical significance was tested with the paired Student’s t test when appropriate.

RESULTS

Clinical and metabolic characteristics of the subjects

The subjects in the troglitazone and metformin groups were matched for age and obesity. After 3 months of troglitazone treatment,
BMI was slightly increased compared with before treatment (P < 0.05). Subjects on metformin for 3 months had no change in BMI. HbA1c, glucose, and insulin concentrations were significantly reduced by troglitazone therapy (Table 1). Triglyceride and free fatty acid concentrations tended to be reduced after troglitazone treatment. Similar to the effects
of troglitazone, metformin treatment significantly decreased HbA1c, glucose, and insulin concentrations (Table 1). Triglycerides were lower (P < 0.05) after metformin treatment (Table 1). The GDR, as determined by a hyperinsulinemic-euglycemic clamp, was increased by 35% (P < 0.01) after troglitazone treatment and by 22% (P < 0.05) after metformin (Table 1). The change in GDR after troglitazone treatment was greater than that seen after metformin treatment (P < 0.05).

Metformin treatment did not alter Akt activity in the basal or insulin-stimulated state (Fig. 2B). The small effect of insulin to stimulate Akt activity before metformin treatment was similar to that in the troglitazone
group before treatment (NS). However, in contrast to troglitazone treatment, there was no enhancement of Akt activation in
response to insulin after metformin treatment (Fig. 2B).

IRS-1, p85, p110β, and Akt1/2 protein levels

The effects of treatment on the expression of key insulin-signaling molecules in skeletal muscle were determined by Western
blotting (Fig. 3). Figure 3 shows representative results from two subjects. Table 2 displays the quantitation of the blots from each group. Neither troglitazone nor metformin treatment had any effect on the
expression of IRS-1, p85, and Akt1/2. However, with troglitazone, but not metformin treatment, the amount of the p110β catalytic
subunit of PI 3-kinase was increased by 46 ± 22% compared with pretreatment (P < 0.05) (Table 2).

DISCUSSION

A growing body of evidence implicates early and intermediate steps in the insulin signaling pathway, including the insulin
receptor, IRS-1, and PI 3-kinase as candidates for defects contributing to insulin resistance in skeletal muscle of diabetic
rodents and humans (7,8,24,25). The present studies were carried out to determine whether the defect in PI 3-kinase activation observed in obese type 2
diabetic people could be reversed by therapy with the insulin-sensitizing agents troglitazone or metformin. The major finding
of this study is that treatment with the TZD troglitazone ameliorates the impairment of insulin-stimulated PI 3-kinase activity
and enhances insulin-stimulated Akt activity in skeletal muscle of obese type 2 diabetic subjects. Unlike troglitazone, the
biguanide metformin does not have these effects. These data suggest that the insulin-sensitizing action of troglitazone, but
not metformin, on whole body glucose disposal in obese type 2 diabetic subjects may be caused, in part, by enhanced PI 3-kinase
activation in skeletal muscle.

In the present study, both troglitazone and metformin treatment were efficacious in reducing plasma glucose levels and increasing
insulin-stimulated GDR (Table 1), consistent with improved insulin sensitivity. Similar results for whole-body glucose homeostasis have been demonstrated
in other studies of type 2 diabetic subjects and obese nondiabetic subjects (26,27), as well as in rodent models of diabetes and insulin resistance (28,29). The antidiabetic actions of troglitazone and metformin appear, however, to be mediated by different mechanisms. The principal
action of troglitazone is to increase insulin-mediated peripheral glucose disposal in skeletal muscle, whereas metformin acts
primarily by decreasing hepatic glucose output (11,16). Although many recent reports have provided new insight into the mechanism of action of the insulin-sensitizing agents,
only a few studies report the effects of TZDs or metformin on insulin action (12,13,21), and there are no data assessing the effects of TZDs or metformin on insulin signaling in skeletal muscle of humans in vivo.

A possible explanation for the enhancement of PI 3-kinase and Akt by troglitazone is that TZD activation of PPAR-γ directly
or indirectly stimulates the activity of these two kinases in skeletal muscle through effects on gene expression. In cultured
muscle cells from obese type 2 diabetic individuals, troglitazone exerts direct effects, increasing PPAR-γ mRNA and protein
levels (30), together with improved insulin-stimulated glucose uptake and glycogen synthase activity (31). In addition, in vitro studies of human adipocytes demonstrate that PPAR-γ activation through TZD directly induces the expression
of genes whose products are involved in insulin signaling (32,33). In this study, we found that the amount of the p110β catalytic subunit of PI 3-kinase was increased with troglitazone treatment,
suggesting the improvement of PI 3-kinase activity could be caused, at least in part, by increased p110β expression in skeletal
muscle. Akt activity but not protein level in muscle is altered by TZD therapy, suggesting that the modulators of Akt activity
may be under transcriptional control of PPAR-γ or that the increased PI3-kinase activity enhances insulin-stimulated Akt activity.
Further data supporting the direct effect of TZDs on insulin signaling include the fact that TZD treatment of human skeletal
muscle cells in vitro induces the activation of PI 3-kinase and Akt (12). Such effects could play an important role in the increased insulin-stimulated glucose uptake and glycogen synthesis that
occurs in these TZD-treated cells (12,31).

In vivo and in vitro studies have demonstrated that chronic hyperglycemia can lead to the development of insulin resistance,
which results from downregulation of glucose transport as well as alteration of insulin signaling in peripheral tissues (34–37). Restoration of euglycemia improves the impaired insulin stimulation of glucose transport in skeletal muscle of type 2 diabetic
subjects (36) and the reduced Akt activity in skeletal muscle of diabetic rats (38). Based on these results, normalization of plasma glucose levels could be expected to lead to an improvement in the insulin-signaling
cascade in type 2 diabetic subjects. Interestingly, we found that troglitazone but not metformin treatment significantly enhances
insulin action at the level of PI 3-kinase and Akt in skeletal muscle. These differences occurred despite similar improvements
in fasting glucose and HbA1c concentrations with the two agents, thus making it unlikely that a generalized reduction in glycemia was responsible for
changes in insulin signaling. Whether the degree of enhancement in insulin activation of PI 3-kinase and Akt in muscle after
troglitazone treatment is sufficient to account for the increased whole-body glucose disposal beyond that attained with metformin
treatment is not known.

Some of the effects of TZDs on insulin signaling may be through secondary mechanisms. Potential factors are plasma fatty acids
and intramuscular lipids because elevations in these parameters are associated with insulin resistance (39,40). As previously reported (41,42), troglitazone treatment tended to lower free fatty acid concentrations (Table 1). TZDs can also reduce accumulation of muscle triglycerides and diacylglycerol (43). Because activation of protein kinase C by elevated diacylglycerol levels in muscle impairs insulin signaling (44), decreases in plasma lipid concentrations with troglitazone treatment could lead to improved insulin signaling by reducing
diacylglycerol in muscle. Troglitazone may also reduce intramyocellular lipid content via PPAR-γ-activated redirection of
free fatty acids from skeletal muscle to storage in adipocyte triglyceride (45). Thus, the ability of troglitazone to improve insulin action in skeletal muscle could involve multiple actions in both adipose
and muscle tissue. Indeed, the increased BMI in our subjects after 3 months of troglitazone treatment may be caused by the
potent adipogenic effect of TZDs (46) or the effect of these agents to promote fluid retention with increased plasma volume (47). Despite increased BMI, these subjects have improved insulin action greater than that in metformin-treated subjects.

Although the main mechanism by which metformin treatment in type 2 diabetic subjects ameliorates insulin resistance is reducing
hepatic glucose production, metformin also increases insulin-stimulated GDRs into skeletal muscle (48,49). In vitro studies demonstrate that high doses of metformin increase insulin-stimulated glucose transport in isolated skeletal
muscle from type 2 diabetic subjects (50). In the current study, despite an increase in the insulin-stimulated glucose disposal rate with metformin therapy, metformin
was unable to enhance insulin activation of PI 3-kinase and Akt in skeletal muscle. In addition, other reports have shown
that the defect of glycogen synthase activity in obese type 2 diabetic subjects was not restored by metformin treatment (51). It is therefore likely that the molecular mechanism whereby metformin exerts its effects on glycemic control is mediated
through other signaling pathways that appear to include activation of AMP kinase (52). This pathway could mediate the effect of metformin to redistribute glucose transporters from an intracellular compartment
to the plasma membrane (53).

In summary, this is the first demonstration that the activities of the insulin-stimulated kinases PI 3-kinase and Akt in human
skeletal muscle are regulated by therapy with insulin-sensitizing agents. Our data demonstrate that troglitazone treatment
improves the impairment of PI 3-kinase activation and enhances Akt activation by insulin in skeletal muscle of obese type
2 diabetic subjects. The change in insulin-stimulated PI 3-kinase activity with troglitazone treatment could be contributed
to, in part, by increased expression of p110β. Although troglitazone is no longer available for the treatment of type 2 diabetes,
the effects on insulin signaling in skeletal muscle are likely to be shared by the TZD class of compounds. In support of this,
our preliminary data show that rosiglitazone treatment also increases insulin-stimulated PI 3-kinase and Akt in some insulin
target tissues of insulin-resistant mice with adipose-specific GLUT4 knock out (data not shown). In contrast, treatment of
diabetic humans with the biguanide metformin has no effect on PI 3-kinase or Akt activation in muscle. Thus, the insulin-sensitizing
effect of the TZD troglitazone in obese type 2 diabetic subjects may be caused, at least in part, by enhancing the insulin-signaling
cascade in skeletal muscle.

IRS-1-associated PI 3-kinase activity in skeletal muscle of diabetic subjects before and after troglitazone (A) or metformin (B) treatment. All subjects underwent a 3-h hyperinsulinemic-euglycemic clamp, and biopsies of vastus lateralis muscle were
performed before and at the end of the clamp. PI 3-kinase activity was measured in IRS-1 immunoprecipitates and was quantitated
using PhosphorImaging. Data are means ± SEM for 6–8 subjects per group. *P < 0.05 vs. no (−) insulin value for each group; †P < 0.05 for post- vs. pretreatment value for insulin-stimulated condition.

Akt1/2 kinase activity in skeletal muscle of diabetic subjects before and after troglitazone (A) or metformin (B) treatment. All subjects underwent a 3-h hyperinsulinemic-euglycemic clamp, and biopsies of vastus lateralis muscle were
performed before and at the end of the clamp. Akt kinase activity was measured in muscle lysates that were subjected to immunoprecipitation
with an antibody that recognizes both Akt1 and Akt2. The immunoprecipitated pellets were assayed for kinase activity using
Crosstide as substrate (10). Each connected set of circles shows values from a single subject. Bars show mean for 6–8 subjects per group. *P < 0.05 vs. the no (−) insulin value for each group; †P < 0.05 for post- vs. pretreatment value for insulin-stimulated condition.

Effect of troglitazone or metformin treatment on insulin-signaling protein levels in skeletal muscle. Skeletal muscle was
obtained before insulin infusion before (pre) or after (post) troglitazone or metformin treatment. Autoradiogram shows representative
Western blots. For p110β, blots for two troglitazone-treated subjects are presented to demonstrate the consistency of the
effect. Quantitation of multiple blots on 7–8 subjects per treatment group is presented in Table 2.

Acknowledgments

This work was supported by a grant from the Medical Research Service, Department of Veterans Affairs, VA San Diego HealthCare
System (VASDHS); the American Diabetes Association; Pfizer Pharmaceuticals; Parke-Davis; grants from the National Institute
of Diabetes and Digestive and Kidney Disease (DK43051 and DK56116); and Grant MO1 RR-00827 from the General Clinical Research
Branch, Division of Research Resources, National Institutes of Health.

We thank Debra Armstrong and Leslie Carter at the VASDHS for assistance with the clamp, biopsy, and assay procedures and Dr.
M.F. White at the Joslin Diabetes Center for the IRS-1 antibody.